The Wiskott-Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin⅐4. Determining how cells regulate actin assembly is vital for understanding motility. Resting cells contain high concentrations of unpolymerized actin, typically 200 -400 M, or about 600 -1200 times greater than the critical concentration for assembly of pure actin (1, 2). Actin-sequestering proteins prevent spontaneous assembly of monomeric actin, and nonmuscle cells contain two such sequestering proteins: the 15-kDa protein profilin and the 5-kDa thymosin-␤4 (3, 4). These proteins were mainly thought to bind actin monomers with a one-to-one stoichiometry and to sequester actin monomers, and both have a higher affinity toward Actin⅐ATP as compared with Actin⅐ADP. The current view is that profilin and thymosin-␤4 are likely to play complementary roles in the cell. Thymosin-␤4 has a single mode of action (5-8), namely binding actin monomers to create a sequestered pool of monomers to supply Actin⅐ATP during periods of active filament growth. The mode of action of profilin, however, is more complex and in many respects has remained controversial and elusive. Although the Profilin⅐Actin complex was initially also thought to be strictly a monomersequestering protein (9 -11), Pollard and Cooper (12) later confirmed the inference that it adds to the barbed ends of actin filaments (13). Initial rate and steady-state assembly measurements showed that profilin can efficiently bind actin monomers (K d Ͻ 5 M) and can weakly interact with filament barbed ends (K capping ϳ 100 M). Profilin catalyzes exchange of free ATP with actin-bound ADP to form actin-bound ATP and free ADP (14 -16). Profilin also exhibits affinity toward oligoproline modules (17), another unique property that facilitates transfer of actin monomers to the polymerization zone (16) lying immediately behind motile Listeria (18), Shigella (19), and vaccinia (20). By concentrating Profilin⅐Actin complex in regions of active filament assembly, explosive rates of filament growth (Ն500 monomers s Ϫ1 ) are readily catalyzed. By contrast, thymosin-␤4 does not bind to oligoproline and fails to concentrate in the polymerization zone.Highly motile cells contain high concentrations of both profilin and thymosin-␤4, and these proteins probably act in concert to promote rapid filament assembly. Pantaloni and Carlier (21) investigated the role of profilin in the absence and presence of thymosin-␤4. They suggested that profilin reduces the critical monomer concentration needed for filament assembly, and they suggested that this is accomplished by accelerating the irreversible hydrolysis of polymer-bound Profilin⅐Actin⅐ATP complex. However, their experimental design was compromised in several significant ways: 1) they used pyrenyl-actin polymerization and had to correct the observed data for the low affinity of profilin for pyrenyl-actin (9,22,23); 2) all of their determinations of the steady-state extent of polymerization rested on th...

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Force generation in several types of cell motility is driven by rapidly elongating cytoskeletal filaments that are persistently tethered at their polymerizing ends to propelled objects. These properties are not easily explained by force-generation models that require free (i.e., untethered) filament ends to fluctuate away from the surface for addition of new monomers. In contrast, filament end-tracking proteins that processively advance on filament ends can facilitate rapid elongation and substantial force generation by persistently tethered filaments. Such processive end-tracking proteins, termed here filament end-tracking motors, maintain possession of filament ends and, like other biomolecular motors, advance by means of 5'-nucleoside triphosphate (NTP) hydrolysis-driven affinity-modulated interactions. On-filament NTP hydrolysis/phosphate release yields substantially more energy than that required for driving steady-state assembly/disassembly of free filament ends (i.e., filament treadmilling), as revealed by an energy inventory on the treadmilling cycle. The kinetic and thermodynamic properties of two simple end-tracking mechanisms (an end-tracking stepping motor and a direct-transfer end-tracking motor) are analyzed to illustrate the advantages of an end-tracking motor over free filament-end elongation, and over passive end-trackers that operate without the benefit of NTP hydrolysis, in terms of generating force, facilitating rapid monomer addition, and maintaining tight possession of the filament ends. We describe an additional cofactor-assisted end-tracking motor to account for suggested roles of cofactors in the affinity-modulated interactions, such as profilin in actin-filament end-tracking motors and EB1 in microtubule end-tracking motors.